Yohei Okada

Affiliation:

1. Department of Physiology, Keio University School of Medicine, Tokyo, Japan2. Department of Neurology and Institute of Neurology, Ruijin Hospital, Aichi Medical University School of Medicine, Aichi, Japan

iPSCs and ESCs are exploited for applications in cell transplantation therapy for regenerative medicine. However, these cells form tumors called teratoma containing differentiated tissues when transplanted into immune-deficient mice. Therefore, the risk of their teratoma-forming tumorigenicity limits their clinical application. Several studies have reported the methods to overcome the risk of teratoma-forming tumorigeniticy (Itakura et al., 2017; Vazquez-Martin et al., 2012). Recently, we reported that iPSCs derived from naked mole-rats lack teratoma-forming tumorigenicity when engrafted into the testes of NOD/SCID mice due to species-specific activation of tumor-suppressor ARF and a disruption mutation of the oncogene ERAS (Miyawaki et al., 2016). In this protocol, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas. This approach can minimize the immune rejection due to the presence of the testicular–blood barrier (Cheng and Mruk, 2012). In addition, this approach is advantageous because transplanted cells are easily identified around the injection site even when they do not form tumors. Thus, the technique described herein is useful for assessing the pluripotency and tumorigenicity of pluripotent stem cells.

At 4, 10, 20, or 28 weeks after transplantation, anesthetize and sacrifice the mice in accordance with the Guide for the Care and Use of Laboratory Animals. Thereafter, dissect the tumors and testes (Figures 2D and 2E).Note: Tumors could be observed 4 weeks and 10 weeks after the injection of mouse iPSCs and human iPSCs, respectively.

Stain sections with hematoxylin and eosin (HE), or subject to immunohistochemical analysis, to detect expression of GFP and differentiation markers. The section of tumors and testes in our previous study are shown in Figure 3 (Miyawaki et al., 2016).

The weights of tumors or testes were analyzed after logarithmic transformation (arbitrary units: 6 + log2). The Bartlett test was used to verify equal variances across populations. The statistical significance of the difference between experimental groups was determined by one-way analysis of variance or the Kruskal Wallis test followed by the Dunn’s method using GraphPad Prism 6 software. Statistical significance was considered when P < 0.05 and all data are displayed as mean ± SEM as in Miyawaki et al., 2016.
Weight of tumors and testes in our previous study are shown in Table 1 for reference (Miyawaki et al., 2016).

This work was supported in part by PRESTO of the Japan Science and Technology Agency, Grants-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Grant-in-Aid for Scientific Research on Innovative Areas ‘Oxygen Biology: a new criterion for integrated understanding of life’ from the MEXT to K.M., and K.M. and S.M. were Research Fellows of the Japanese Society for the Promotion of Science.